Catenulisporolides, Glycosylated Triene Macrolides from the

Letter Cite This: Org. Lett. 2018, 20, 7234−7238

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Catenulisporolides, Glycosylated Triene Macrolides from the Chemically Underexploited Actinomycete Catenulispora Species Sangkeun Son,† Young-Soo Hong,†,‡ Yushi Futamura,∥ Mina Jang,†,‡ Jae Kyoung Lee,† Kyung Taek Heo,†,‡ Sung-Kyun Ko,†,‡ Jung Sook Lee,§ Shunji Takahashi,⊥ Hiroyuki Osada,∥ Jae-Hyuk Jang,*,†,‡ and Jong Seog Ahn*,†,‡ †

Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Korea Department of Biomolecular Science, KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon 34113, Korea § Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Korea ∥ Chemical Biology Research Group, RIKEN Center for Sustainable Research Science, Saitama 351-0198, Japan ⊥ RIKEN-KRIBB Joint Research Unit, RIKEN Center for Sustainable Research Science, Saitama 351-0198, Japan

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‡

S Supporting Information *

ABSTRACT: New glycosylated 26-membered triene macrolides catenulisporolides, the first polyketide metabolites from Catenulispora species, were obtained by targeting slow-forming colonies on selection agar plates and applying long-term cultivation. Their structures, including the full stereochemistry, were defined by comprehensive spectroscopic and chemical methods and confirmed by bioinformatics analysis. Analysis of the genome sequence revealed the responsible biosynthetic gene cluster spanning ∼160 kbp, and feeding experiments with isotope-labeled precursors showed that isovaleric acid acts as a rare starter unit. Catenulisporolides exhibited antimalarial activities against resistant strains of Plasmodium falciparum, and enhanced activity was observed in semisynthetic derivatives.

N

To obtain actinomycetes that are difficult to isolate, we conducted a long-term incubation of the soil suspension on agar plates and selectively picked up slow-forming colonies. Indeed, 16S rRNA gene sequence analysis of the isolated strains that appeared after a 7-week incubation indicated that they belong to underexplored genera, including Catenulispora and Actinoallomurus. Given their slow growth under laboratory culturing conditions, we monitored the production of secondary metabolites by LC-MS over longer periods (up to 20 days) compared to the usual conditions (Figure S1). This analysis revealed that strain KCB13F217 belonging to the genus Catenulispora started to produce a possibly new triene metabolite with a molecular mass of 1,116 Da after 8 days of cultivation. The production of the metabolite was dramatically increased after 14 days, indicating that its production is highly dependent on the culture period. The genus Catenulispora is one of the least explored actinomycete genera in terms of novel secondary metabolite discovery. It was first described in 2006, and only two classes of metabolites, lantipeptide catenulipeptin and aminocoumarin cacibiocins, both of which were isolated from the Catenulispora acidiphila type strain, have been characterized.4 As the highest yields of structurally related minor congeners were observed in a culture with glycerol-

atural products from actinomycetes, especially those from the largest genus of Actinobacteria Streptomyces, have long been renowned as a prime source of bioactive compounds with enormous chemical diversity.1 Although exploring novel natural products from Streptomyces is still valuable, screening common actinomycetes often leads to the rediscovery of already known compounds.2 Rare actinomycetes, whose isolation frequency is much lower than that of Streptomyces, have increasingly gained attention as promising sources of new natural products. In fact, many of the clinically important compounds have been isolated from rare actinomycetes, encouraging intensive chemical studies on yet unexplored genera.3 However, systematic chemical investigations on novel actinomycete groups are rather difficult, primarily due to the inability to successfully isolate the underexplored group of actinomycetes and having them produce substantial amounts of secondary metabolites for bioassays and structural determination. In this study, we hypothesized that hard-to-reach actinomycetes grow slowly under laboratory culture conditions, and this makes it difficult for natural product chemists to unveil their biosynthetic potential. It was therefore expected that targeting slow-growing colonies on selection agar plates and culturing them over a longer period can rationally increase the opportunities for the identification of untapped actinomycetes and their secondary metabolites. © 2018 American Chemical Society

Received: October 3, 2018 Published: October 31, 2018 7234

DOI: 10.1021/acs.orglett.8b03160 Org. Lett. 2018, 20, 7234−7238

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Organic Letters based GLY medium on the 15th day, this condition was selected for the large-scale culture. Four compounds named catenulisporolides A−D (1−4) were further purified by solvent extraction and LC-MS-targeted purification (Figure 1).

Figure 2. Selected 2D NMR correlations of 1.

oleandrose (Ole) and O-β-olivopyranosyl-(1 → 3)-O-βolivopyranose (Oliv 1 and 2) and connected to C-15 and C31, respectively (Figures S9 and S10). Catenulisporolide A (1) contains 26 stereocenters, 14 of which reside in the aglycone. The large 3JH‑10,H‑11 (10.0 Hz) in combination with the intense ROESY correlations (9-OH/H11/H-13) indicated a trans 1,2-diaxial relationship between 9OH/H-10/H-11, thus confirming the relative configuration of the tetrahydropyran ring moiety (Figure 2B). The relative configuration of the chiral centers present in the macrolactone portion and acyclic chain was determined by applying the Jbased configuration analysis (JBCA) and Kishi’s universal NMR database. To observe the weight average of the coupling constants from the flexible acyclic systems, compound 1 was first linearized by methanolysis with NaOMe to 1a. A series of homo- and heteronuclear coupling constants of 1a measured through a combination of DQF-COSY, HETLOC, and HECADE experiments using CD3OD enabled the assignment of the relative configuration for C-13 to C-17 (Figure S5). The coupling constants from the crowded oxygenated methine and methylene regions could be resolved with high-field 2D NMR spectroscopy (900 MHz). The relative stereochemistry of the C-25 to C-31 acyclic segment was established by the JBCA of 1 (Figure S6). We used CD3COCD3 for those of C-25 to C-31 because no measurable difference in the chemical shift between the diastereotopic methylene protons at C-26 could be observed from 1 in CD3OD and DMSO. To further support the relative configuration of the C-27 to C-30 portion, two contiguous propionate units were analyzed by applying Kishi’s universal NMR database.5 The 13C NMR chemical shifts observed for C-28 and C-37 were compared with those of each diastereomer in the database. This approach predicted the αααβ configuration (Figure S7), which is in accordance with the result of the JBCA. Next, the absolute D configurations of the sugar units were determined by chromatographic comparison of thiocarbamoylthiazolidine derivatives of the sugar in the acid hydrolysate of 1 with those of the authentic standards which contain D-olivose and L-oleandrose, respectively (Figure S8).6 The absolute configuration of the aglycone was then derived from the ROESY correlations between the protons in the sugar units and the aglycone. The ROESY correlations (H-1′/H-15; H2a′/H-17, H-19) confirmed the spatial configuration of C-1′ and enabled the stereochemical assignment of C-15 as R (Figure 2B). In the same manner, the stereochemistry of C-31

Figure 1. Chemical structures of catenulisporolides A−D (1−4) from Catenulispora sp. KCB13F217 and the semisynthetic derivatives.

Catenulisporolide A (1), isolated as a white amorphous solid, was assigned the molecular formula C58H100O20 by a sodium adduct ion (m/z 1139.6694 [M + Na]+) on HRESIMS combined with the NMR data. When the methanol solution of 1 was exposed to room light for 5 days, it was found to be stable, and only a very tiny amount of 1 was converted to the O-methylated derivative (Figure S2). Structural elucidation of the 26-membered triene aglycone moiety of 1 was performed by detailed analysis of 1D and 2D NMR data (see the Supporting Information (SI) for detailed structural elucidation). Interpretation of HSQC-TOCSY spectra was crucial for confident assignment of the completely overlapped signals of C-26 and C-30 at δC 38.2 (Figure 2A). According to the strong ROESY correlations of the syn 1,3-diaxial protons and 3JH,H coupling constants from the DQF-COSY data, together with HMBC correlations, the sugar units were established as β7235

DOI: 10.1021/acs.orglett.8b03160 Org. Lett. 2018, 20, 7234−7238

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Organic Letters was determined as S by the ROESY correlations (H-1″/H-31; Ha-2″/H3-38; H-5″/H3-39). Hence, the absolute configurations of all the chiral centers present in the aglycone, except for C-7, were established as 9S, 10R, 11S, 13S, 15R, 17S, 24S, 25R, 27R, 28S, 29R, 30R, and 31S. To determine the absolute configuration at C-7, 1 was treated with NaOMe to yield the linearized product 1a, which was subjected to ozonolysis and a reductive workup. The resulting reaction mixture was then successively treated with (R)- or (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl), which yielded a complex mixture of Mosher derivatives. Careful HPLC separation yielded tetra-(S)- and (R)-MTPA esters (1b and 1c) of the C-1 to C-18 segment, and analysis of the ΔδH values (δS−δR) enabled the assignment of the R configuration at the C-7 position (Figure 3). The

The structure of catenulisporolides suggests a polyketide origin with chain assembly by a type I polyketide synthase (PKS). To address the biosynthetic gene cluster for the catenulisporolides, the genome sequence of Catenulispora sp. KCB13F217 (12.1 Mbp) was analyzed using the antiSMASH 4.0 software.7 According to the prediction (Table S1) in combination with a retrosynthetic approach, the region responsible for catenulisporolide (cat) gene cluster which contains five type I PKS and sugar biosynthetic genes was proposed (Figures 4 and S12; Tables S1, S2, and S3). The cat gene organization was unusual in that catA gene coding for the loading module and the first extender module is located ∼66 kbp upstream of the other PKS genes. The similar characteristic of the gene organization was observed in the milbemycin biosynthetic gene cluster.8 The loading module of the CatA was found to lack an intact cognate AT domain. The biosynthesis of the catenulisporolides begins with the loading of a branched side chain as a starter unit, suggesting that isovaleryl-CoA could be involved in the biosynthesis. This starter unit is rare among macrolide compounds with one example in the sulfangolid family, sulfate ester containing macrolides from myxobacteria.9 If this is the case, isovaleryl-CoA might be derived from the L-leucine,10 and loaded by a discrete AT enzyme encoded in cat55 or outside the cluster.11 By the consecutive action of transaminase and the branched chain keto acid dehydrogenase complex, Lleucine undergoes transamination to 4-methyl-oxovaleric acid, which is further processed into isovaleryl-CoA. On the other hand, the BLASTP and phylogenetic analysis indicated that Cat34 and Cat53 are classified in the fatty acyl-AMP ligase (FAAL) clade (Figure S13). FAAL was previously characterized to introduce long-chain fatty acids in the biosynthesis of polyketides and nonribosomal peptides,12 suggesting the possibility that the biosynthesis of catenulisporolides starts when FAAL activates isovaleric acid as the acyl-adenylate form and then loads it onto the ACP in module 1 (Figure S14).13 Because genetic manipulation was not achieved for the genus Catenulispora due to slow growth and the lack of a gene transformation system, feeding studies using isotope labeled Lleucine-d3, 4-methyl-2-oxovaleric acid-d3, and isovaleric acid-d9

Figure 3. Preparation of tetra-(S)- and (R)-MTPA esters of the C-1 to C-18 segment of 1.

established stereochemistry of C-11, C-17, and C-4′ based on Mosher’s method was consistent with the result of the JBCA and sugar analyses. It is worth mentioning that the JBCA of 1 yielded an incorrect configuration for C-13 to C-17 which is not matched with the result of Mosher’s analysis (Figure S9), indicating that it is not desirable to apply the JBCA method for small-ring macrolides possessing a steric structure, and a combination of various approaches is necessary for unambiguous stereochemical determination of macrolides.

Figure 4. Proposed model of catenulisporolide biosynthesis. (A) Organization of the catenulisporolide biosynthetic gene cluster (accession number MH844630). (B) Module and domain organization of the PKSs involved in the biosynthesis of catenulisporolide (AT, acyl transferase; ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; TE, thioesterase). 7236

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Therefore, this result provides a valuable empirical example which validates the bioinformatics approach as a reliable method for stereochemical assignment. Catenulisporolides were first subjected to antibacterial, antifungal, and cytotoxic assays; however, they turned out to be inactive up to 30 μM. We next evaluated their antimalarial potential against Plasmodium falciparum. In the antimalarial assay against P. falciparum strains including 3D7 and two chloroquine resistant strains Dd2 and K1, catenulisporolides inhibited the growth of parasites (Table 1), and 4 was found to

were performed to examine the biosynthetic origin of a starter unit. LC-MS analysis revealed that mass shifts were observable from all three compounds. Of the compounds tested, isovaleric acid-d9 was the most efficiently utilized for the biosynthesis of 1 (Figure S15). In addition, the 1H NMR data of the labeled 1 obtained from an isovaleric acid-d9 feeding experiment indicated the signal intensities of H3-34 and H3-39 significantly decreased to ca. 50% of the unlabeled natural 1 (Figure S16). The relatively low incorporation of L-leucine-d3 and 4-methyl2-oxovaleric acid-d3 is likely due to their conversion into isovaleric acid-d3 during leucine catabolism. Although feeding experiments could not rule out the possibility that the starter unit originates from both the acyl-CoA and acyl-adenylate forms, they revealed that the carboxylic acid substrate acts as the starter unit for the biosynthesis of catenulisporolides. This deduction was further supported by the high starter unit selectivity. Loading modules consisting of AT and ACP domains that act on branched acyl-CoA tend to recognize a diverse set of starter units.14 We tested incorporation of other starter units into catenulisporolides by feeding different shortchain carboxylic acids and branched amino acids; however, none of the expected products were produced (Figure S17), supporting the possibility that FAAL with a substrate preference for isovaleric acid is involved in the initiation of the biosynthesis. The predicted substrate specificity for each extending domain was in good agreement with the structure of aglycone in 1. One possible explanation for the specific dehydration of the C-11 hydroxy group in 4 would be that the DH domain of module 12 is active on the preceding module 11 and partially dehydrates the hydroxy group on C-11 (see the SI for detailed structural elucidation of 2−4). This might result in an altered spatial orientation of the C-9 carbonyl, thus preventing the formation of the hemiketal ring. The unusual double bond formation by the DH domain of the downstream module has been proposed for several myxobacterial metabolites, and experimental evidence was provided for the epothilone gene cluster.15 To further demonstrate the absolute configurations of catenulisporolides, KR sequences were aligned and divided into A or B type based on the characteristic motif of each group (Figure 5).16 For the stereocenters bearing a methyl

Table 1. Antimalarial Activity of Catenulisporolides and the Semisynthetic Derivatives IC50 (μM) Plasmodium falciparum

a b

mammalian cells

compds

3D7

Dd2

K1

NRK

WI-38

1 2 3 4 1a 4a 1d 1e Chloroquine

18 19 >30 2.9 16 5.5 1.7 1.8 0.055

15 21 23 2.8 14 5.6 1.3 1.5 0.64

14 18 26 2.1 13 4.4 1.0 1.4 1.7

>100 >100 >100 33 >100 >30a 10 6.8 n.t.b

>100 >100 >100 56 >100 >30a 10 14 n.t.b

Compound 4a was only tested up to 30 μM due to a lack of material. n.t.: not tested.

be the most active. These results prompted us to further examine the antimalarial activities of semisynthetic derivatives prepared by chemical derivatization procedures (Figure 1). The linearized derivatives 1a and 4a still exhibited inhibitory activities. Therefore, the macrolactone scaffold appears not to be a requisite for the antimalarial activities. The dehydrated derivatives 1d and 1e, prepared by treatment of 1 with ptoluenesulfonic acid (Figure S11), exhibited the most potent activities, especially against the K1 strain. Compared with the MIC values against normal mammalian cells, all tested compounds showed 5−10 times lower MIC values against P. falciparum, indicating a decent selectivity for malaria. Catenulisporolides represent the first example of macrolide metabolites containing a 26-membered triene aglycone, and also feature the isovaleric acid starter unit which has been rarely encountered among macrolides. These characteristic structural features might explain the bioactivities of catenulisporolides which differ from the structurally relevant toxic macrolides.17 As can be seen from a large biosynthetic gene cluster for catenulisporolides, which is difficult to be rigorously characterized by metagenome mining, our findings underline that continued and careful efforts for isolation and cultivation are crucial for exploring undiscovered microbial natural products, and extending the incubation and cultivation period is a simple, but considerably important factor that should not be underestimated in the study of hard-to-reach microorganisms. Elucidating the biosynthetic mechanisms, especially for the loading of the unusual starter unit, and activating the expression of the cryptic gene clusters in Catenulispora sp. KCB13F217 are important future challenges that should greatly enhance our understanding of secondary metabolites from novel actinomycete groups.

Figure 5. Stereochemical determination of the aglycone moiety. (A) Absolute configurations determined by chemical and spectroscopic methods. (B) Bioinformatics-based configurational assignment.

group derived from 2-methyl-branched substrates (methylmalonyl-CoA), the KR domains were further divided into four subgroups according to the presence of the residue indicative of the α-methyl epimerizing activity. This consequently enabled the prediction of the absolute configurations for 13 chiral centers which matched perfectly with those defined based on chemical and spectroscopic analyses (Figure 5A). 7237

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03160.

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Detailed experimental procedures and tables along with figures for the NMR and MS spectra and the feeding experiments (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.-H.J.). *E-mail: [email protected] (J.S.A.). ORCID

Young-Soo Hong: 0000-0001-7768-2239 Hiroyuki Osada: 0000-0002-3606-4925 Jae-Hyuk Jang: 0000-0002-4363-4252 Jong Seog Ahn: 0000-0001-5166-4358 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Harumi Aono (RIKEN CSRS) for the biological activity tests. This work was supported by an International Joint Research Project (ASIA-16-011) of the NST (National Research Council of Science & Technology), Young Researcher Program (NRF-2017R1C1B2002602) of the NRF (National Research Foundation of Korea), and the KRIBB Research Initiative Program funded by the Ministry of Science ICT (MSIT) of the Republic of Korea. We also thank the Korea Basic Science Institute, Ochang, Korea, for providing the NMR and HRESIMS data.

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REFERENCES

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DOI: 10.1021/acs.orglett.8b03160 Org. Lett. 2018, 20, 7234−7238